MICROBIAL CONTROL

AND
IDENTIFICATION

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Microbial Control
and Identification

Strategies
Methods
Applications

Dona Reber and Mary Griffin

Editors

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PDA
Bethesda, MD, USA
DHI Publishing, LLC
River Grove, IL, USA

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10 9 8 7 6 5 4 3 2 1

ISBN: 978-1-942911-27-2
Copyright © 2018 Dona Reber and Mary Griffin
All rights reserved.

All rights reserved. This book is protected by copyright. No part of it may

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Where a product trademark, registration mark, or other protected
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While every effort has been made by the publisher and the authors to
ensure the accuracy of the information expressed in this book, the
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expressed in this book are those of the authors and may not represent those
of either Davis Healthcare International or the PDA, its officers, or directors.

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We dedicate this book to Cynthia Sarnoski, Ph.D., our long-time

mentor and advisor who offered invaluable encouragement and
support throughout the writing and editing of this book.

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CONTENTS

Foreword xiii
Preface xv
About the Editors xvii

SECTION 1 – STRATEGIES
1 REGULATORY AND COMPENDIAL
GUIDANCE ON MICROBIAL IDENTIFICATION 3
Tony Cundell
Introduction 3
Regulatory Guidelines 5
Compendial Guidelines 7
Detecting and Identifying Fungal Isolates from
Manufacturing Facilities 10
Human Microbiome 14
Panel-based Molecular Diganostics 16
Strain Typing 17
Whole Genomic Analysis for the Detection
and Identification of Microbial Pathogens 18
Conclusions 18
References 18
About the Author 22

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vi Microbial Control and Identification

2 MICROORGANISM IDENTIFICATION AND

ITS ROLE IN CONTAMINATION CONTROL 23
Jeanne Moldenhauer
Introduction 23
Background 24
Identification Methods 25
How Does the Organism Identification Aid in Finding
a Root Cause for Contamination? 28
Conclusion 39
References 39
About the Author 41

3 MEANINGFUL CONTAMINATION
CONTROL TRENDING IN
BIOLOGICAL MANUFACTURING 43
Ren-Yo Forng and Jeanne Mateffy
Purpose 43
Trending 45
Process Monitoring 58
Conclusion 65
Case Study 66
References 68
About the Authors 70

4 ASSESSMENT OF MICROBIAL IMPACT

FROM A LOW BIOBURDEN PERSPECTIVE 73
Hilary Chan and Lynn Johnson
Introduction 73
Microbial Impact and Significance for Patient Safety
and Product Quality 78
Microbial Impact Assessment Considerations 81
Microbial Impact Assessment Tools 91
Lifecycle Management and Quality by Design 106
Summary 108
References 109
About the Authors 115

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Contents vii

5 QUALITY RISK MANAGEMENT IN THE

CONTEXT OF VIRAL CONTAMINATION 117
Consortium on Adventitious Agent Contamination in
Biomanufacturing (CAACB)
Introduction 117
Use of QRM Tools 124
Initiation of Risk Management Process 146
Risk Assessment 149
Risk Control 153
Result of Risk Management Process 156
Risk Review 161
References 161
About the CAACB 163

6 TRENDING OF MICROBIOLOGICAL
ISOLATES IN THE BIOPHARMACEUTICAL
ENVIRONMENT 165
Dona Reber
Regulations and Guidelines 166
Uses for EM Trending 168
EM Sample Types of Monitoring Trended 172
Trending Frequency 172
Microbial Identification Strategy 174
Steps in Isolate Trending 176
Conclusion 191
References 191
About the Author 194

7 BURKHOLDERIA CEPACIA COMPLEX:

CHARACTERISTICS, PRODUCT RISK,
AND TESTING ASSESSMENTS 197
Tim Sandle
Introduction 197
Organism Characteristics 199
Origins in Pharmaceutical and Healthcare 200
Potential Risk to Patients 203
FDA Alert and the Testing Debate 204
Testing Scope 212

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viii Microbial Control and Identification

Tests for BCC 212
Assessment of Test Data 216
Summary 219
References 220
About the Author 228

SECTION 2 – METHODOLOGIES
8 OVERVIEW OF CONVENTIONAL AND
EMERGING MICROBIAL IDENTIFICATION
METHODS 231
Frank E. Matos and Jennifer R. Reyes
Introduction 231
Microbial Identification: What Method to Use 232
Isolation of Microorganisms in Pure Culture 234
Methods for Characterization and Preliminary Screening 235
Phenotypic Methods for Microbial Identification 241
Fatty Acid Analysis Using the Midi Sherlock® 246
MALDI-TOF 248
Genotypic Methods for Bacterial Identification 249
Ribotyping 254
Identification of Fungi 258
Molecular Methods for Fungal Identification 265
Use of Contract Laboratories for Microbial Identification 268
Conclusion 269
References 269
About the Authors 273

9 QUALIFICATION AND IMPLEMENTATION

OF MALDI-TOF TECHNOLOGY FOR USE
IN A QUALITY CONTROL LABORATORY 275
Jennifer R. Reyes, Frank E. Matos and Gary Kruppa
Introduction: Microbial Identification Using MALDI-TOF
Mass Spectrometry 276
In-House Evaluation 285
Qualification Activities 287
Improvements 293
Routine Use Activities 295
References 298
About the Authors 301

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Contents ix

10 TESTING FOR MYCOPLASMA

IN BIOTHERAPEUTICS 303
Houman Dehghani and Martina Kopp
Introduction 303
General Biology of Mycoplasma 304
Regulatory Guidelines 306
Traditional Testing Methods for Mycoplasmas 307
Emerging Rapid Testing Methods for Detection
of Mycoplasma 309
Conclusion 315
References 315
About the Authors 317

11 VIRUS DETECTION AND TESTING METHODS 319

Cassandra L. Braxton and Maria M. Bednar
Introduction 319
Key Elements 320
Adventitious Virus Detection 323
New Rapid Molecular Methods 328
Advanced Virus Detection Using NGS 330
Challenges of NGS in a Biopharmaceutical Setting 346
NGS Regulatory Guidance 349
Conclusion 351
References 352
About the Authors 360

12 DATA INTEGRITY IN THE LABORATORY 361

Jeanne Moldenhauer
Introduction 362
What are Data? 363
Requirements for Data Integrity 363
Data Pedigree 364
Regulatory Expectations for Data Integrity 365
Difficulties in Having Complete Data 366
Evaluating Data Integrity in the Laboratory 367
Triggers for Checking Data Integrity in the Laboratory 369
The Effect of Personnel Behavior on Data Integrity 375
Conclusion 376
References 376
About the Author 377

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x Microbial Control and Identification

SECTION 3 – APPLICATIONS
13 DISINFECTANT PERFORMANCE AGAINST
FUNGI,VIRUSES AND BACTERIA 381
James Polarine, Jr., David Shields, and Jennifer Loughman
Introduction 381
Disinfectant and Sporicide Activity against Fungal Spores 382
Disinfectant and Sporicide Activity against Viruses 391
Disinfectant and Sporicide Activity against Bacteria 393
Conclusion 400
References 401
About the Authors 403

14 ENVIRONMENTAL ISOLATES IN
PHARMACEUTICAL MANUFACTURING 405
Angel L. Salamán-Byron
Introduction 405
Regulatory and Compendia Guidance 406
Evaluation and Selection of EIs 409
Application EIs in Pharmaceutical Test Methods 421
Conclusion 426
References 427
About the Author 438

15 MANAGING STOCK CULTURES 439

Vanessa Vasadi-Figueroa
Introduction 439
Applications and Use 440
Top Six Lineup 441
Culture Libraries 442
Handling 443
Storage Methods 444
Cryopreservation 446
Lyophilization 448
Culture Recovery 449
Market Availabilities 452
Documentation and Inventory Management 454
Conclusion 454
References 455
About the Author 456

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Contents xi

16 COMPETENCY-BASED TRAINING APPROACH

FOR MICROBIAL IDENTIFICATIONS 457
Edward S. Balkovic and Gary C. du Moulin
Scope of Microbiology Based Training 461
Expectations for Establishing the Training Program 462
Expectations for Those Participating in the Training Program 466
Training Material Development 469
Training Phase – Process 474
Training Phase – Task Specific Considerations 478
Assessment Phase 484
Summary 486
References 487
About the Authors 491

17 MOLD CONTAMINATION IN THE

PHARMACEUTICAL INDUSTRY 493
Brian G. Hubka and Jeanne Moldenhauer
What is Mold? 493
Why do I Care About Mold? 494
Criteria for Mold Growth 499
Mold Detection and Identification 501
Setting Limits for Mold Contamination 509
Mold Remediation Methods 511
Mold Prevention Methods 514
References 517
About the Authors 520

18 KEY FEATURES OF A BIOSAFETY PROGRAM

FOR THE BIOPHARMACEUTICAL INDUSTRY 523
Jessica Avizinis
Relevance of Biosafety to Drug Development,
Manufacturing and Testing Operations 524
The Importance of Biosafety: Impacts of Exposure 527
Key Features of a Biosafety Program 530
Future Biopharmaceutical Biosafety – Special Challenges 560
Conclusion 561
References 562
About the Author 567

Index 569

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FOREWORD

Many years have gone by since I first dabbled in microbiology,

specifically as it relates to the manufacture of medical products and
the impact microbiology has upon the pharmaceutical industry
worldwide. I would have greatly appreciated being better versed
back in those days with Strategies, Methods, Applications, all topics
that remain to be within our focus and scientific realm of interest.

The book’s stable of authors are subject matter experts, world

renowned and highly respected microbiologists in their respective
field of science. That said, you will appreciate reading their depth
and breadth of experience regarding current microbiological
challenges. For example, Cundell, Moldenhauer, Mateffy and
Sandle provide their perspectives on strategies that you will find
worthy of your consideration.

The microbiological control and identification as well as

microbiological assessment are areas that we all, and quite rightly
so, continue to focus our energies and resources. Part and parcel
with microbial identification includes trending of the microbial data
and corrective measures that may need to be implemented to
preclude and/or reduce the presence of microbial and bacterial
endotoxin contamination. We all appreciate an inability to escape

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xiv Microbial Control and Identification

from having a scientific discussion without including some form of

risk assessment and risk management, which is no less important
with regards to viral inactivation. I dare say you will find the
chapter worthy of your read.

The rapid microbiological methods (e.g., MicroSEQ®, MALDI-

TOF) are more common place in todays’ laboratories and a far cry
from someone who used to prepare a sea of the biochemicals,
micro-bial test reagents, poured a river of agar plates and yes,
indeed I washed my share of test tubes. The chapter includes
laboratory equipment qualification considerations that historically
may have not been performed by the laboratory staff. The reader
will find it rewarding as they come to better understand and better
appreciate how the equipment qualification can impact the
microbiological results.

There is a subject that has captured much of our time and to a

notable extent has stretched precious resources. Jeanne Moldenhauer
provides welcomed guidance and perspectives on this subject, Data
Integrity, which you will find worthy of consideration to address and
implement the necessary measures to preclude the accidental or
unintentional deletion or omission of microbiological data.

I continue to assess how well a company understands the

microbiological impacts upon the manufacturing processes.
Irrespective of whether the commodity is sterile or non-sterile, a
comprehensive microbiological assessment is value added to any
manufacturing operation. Equally important, which all have heard
me say throughout the years, did the comprehensive assessments
include a Microbiologist at the discussion table during the
evaluation and implementation process. The individual topics
within the text are of welcomed value to any organization. That
said, when considering the Strategy, Methods and Applications,
collectively, I would point out that is not unlike how I might possibly
assess a medical products manufacturer.
Thomas J. Arista
June 14, 2018

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PREFACE

It has been six years since we edited our initial volume on Microbial
Identifications: The Keys to a Successful Program for the PDA and DHI
Publishing, LLC. Microbial identification continues to evolve with
new technologies and expectations, yet remains so fundamental to
microbial control of quality biopharmaceutical products.

We decided it was the perfect time to once again bring together

a group of expert microbiologists and biopharmaceutical industry
leaders to discuss microbial identifications in a new light, that of
how microbial identification knowledge is a cornerstone in the
concept of microbial and contamination control programs. The
chapters show how microbial control programs for our facilities,
equipment and personnel result in a positive impact on our
products and ultimately our patients.

Some of these authors were included in our initial volume and

we welcome them back for their continued support and excellent
and informative chapters: Drs. Anthony M. Cundell, Jeanne Molden-
hauer, Martina Kopp and Houman Dehghani. The remaining
authors are new for us, but not to our industry by any means – all are

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xvi Microbial Control and Identification

well known for their expertise, and we are very pleased that they
have joined us here. Please open the book and you will most likely
recognize some of these authors, others are newcomers to writing
chapters, and we are delighted they all rose to the challenge.

This book has three sections: Strategies, Methods and

Applications. Strategies cover regulations and regulatory
expectations as well as strategies for trending, risk assessments, and
risk management. Methods include current best practice
microorganism identification methods, both conventional and
emerging rapid methods for bacteria, viruses, mycoplasma and
fungi. Applications include microbiology laboratory training for
identifications, use of environmental and control microorganisms,
disinfectant effectiveness and best practices, and an up and coming
chapter subject, biosafety for laboratories, manufacturing facilities
and personnel.

It is recognized that each author has taken time from his or her
very busy schedules and free time because of a passion for
microbiology and microbial control, and with it the desire to share
knowledge with others in our industry. This is very much
appreciated by us. We also share this passion and dedication as
evidenced by our many years of working and presenting on
microbiological issues.

This text will provide valuable information for the new micro-
biologist as well as a reference for well-seasoned professionals. We
sincerely hope you enjoy the book.

Dona Reber and Mary Griffin

June 2018

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ABOUT THE EDITORS

Dona Reber has more than 25 years of academic and industrial

microbiology experience, including developing and implementing
contamination control strategies for aseptic/non aseptic manu-
facturing, microbiology testing/development, and managing/
networking microbiology laboratories. She is Senior Manager of
Microbiology and Aseptic Support, Global Quality Operations for
Pfizer, responsible for assessing aseptic processing facilities,
investigations, troubleshooting, and developing policy documents
for microbiology.

Dona holds B.S. and M.S. degrees in related sciences, is (SM)

NCRM certified, has been a PDA member for 20+ years, and a
longtime chair of the Pharmaceutical Micro Expert Discussion
Group. She has numerous publications on microbial identifications
systems, environmental monitoring and microbiology risk
assessments. She has co-authored three PDA Technical Reports:
TR#13, Fundamentals of an Environmental Monitoring Program;
TR#67, Exclusion of Objectionable Microorganisms from Non
Sterile Pharmaceutical, Medical Devices and Cosmetics; and TR#70,

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xviii Microbial Control and Identification

Fundamentals of Cleaning and Disinfection. She is on a PDA Task

Force for Low Bioburden Environmental Monitoring. She has
participated in instructing new FDA inspectors, and is an instructor
for the PDA on TR#13, Fundamentals of an EM Program. She
presented at the most recent PDA Micro Conference on
environmental monitoring for low bioburden processes.

Mary Griffin, Principal, MG Quality Microbiology Consulting,

LLC, is a subject matter expert in microbiology with more than 35
years of academic (University of Michigan; University of
Massachusetts) and global biopharmaceutical experience.

She initiated and directed the Quality Control Microbial Science

and Technology department at Pfizer (previously Wyeth, Andover,
MA) during a period of significant growth. She adapted her
extensive knowledge of microorganisms and classical identification
techniques to successfully implement both identification and rapid
automated detection systems to keep the facility on the cutting edge
of novel technologies.

She continues to support the industry as a consultant through

the application of her microbiological identification and rapid
technologies expertise for microbial control. She holds a M.S. degree
in Microbiology and is certified as a Specialist Microbiologist SM
(NRCM). She is an active member of the Parenteral Drug
Association (New England Chapter) and is published in the
pharmaceutical industry.

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